Sialyltransferase variants having neosialidase activity

Patent metadata

Field	Value
Publication number	US-11739305-B2
Application number	US-201816955737-A
Country	US
Kind code	B2
Filing date	Dec 21, 2018
Priority date	Dec 21, 2017
Publication date	Aug 29, 2023
Grant date	Aug 29, 2023

Abstract

α2-6-Sialyltransferase (2,6ST) variants having improved α2-6-specific sialidase activity as compared to the native 2,6ST enzymes are described. The variants include GT80 sialyltransferases such as P. damselae Pd2,6ST. Methods for making de-sialylated products and screening sialidase activity are also described.

First claim

What is claimed is:

1. An α2-6-sialyltransferase (2,6ST) variant having increased α2-6-specific sialidase catalytic efficiency (k cat /K M ) as compared to a native 2,6ST, wherein the 2,6ST variant comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2 and comprising one or more of:
a leucine residue at a position corresponding to residue 232 of SEQ ID NO: 1;
a serine residue at a position corresponding to residue 356 of SEQ ID NO: 1; and
a phenylalanine residue at a position corresponding to residue 361 of SEQ ID NO: 1.

2. The 2,6ST variant of claim 1 , wherein the 2,6ST variant comprises:
the leucine residue at the position corresponding to residue 232 of SEQ ID NO: 1;
the serine residue at the position corresponding to residue 356 of SEQ ID NO: 1; and
the phenylalanine residue at the position corresponding to residue 361 of SEQ ID NO: 1.

3. The 2,6ST variant of claim 1 , wherein the 2,6ST variant comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.

4. The 2,6ST variant of claim 1 , wherein the 2,6ST variant comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:2.

5. An isolated nucleic acid encoding the 2,6ST variant according to claim 1 .

6. A vector comprising the nucleic acid of claim 5 .

7. An Escherichia coli host cell comprising the nucleic acid of claim 5 .

Assignees

• Univ California

Inventors

• Chen Xi
• Mcarthur John B
• Fisher Andrew J

Classifications

• C12N9/1081 Primary

  transferring other glycosyl groups (2.4.99) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12Y204/99001

  Beta-galactoside alpha-2,6-sialyltransferase (2.4.99.1) · CPC title

• C12Y204/99004

  Beta-galactoside alpha-2,3-sialyltransferase (2.4.99.4) · CPC title

• C12P19/04 Primary

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title